DE2402221A1 - PROCESS FOR THE PRODUCTION OF HESPERETINE DIHYDROCHALCONGLUCOSIDE - Google Patents

Description

L. Givaudan & Qe Societe Anonyme, Vernier-Geneve (Switzerland)

Process for the preparation of hesperetin dihydrochalcone glucoside

It is known that the tasteless G-lycoside hesperidin, which occurs mainly in the peel of citrus fruits, by treatment with alkali, hydrogenation and enzymatic hydrolysis into the very sweet hesperetin dihydrochalcone glucoside, which can therefore be used as a sweetener the formula

CH ₂ OH

can be transferred (see e.g. U.S. Pat. No. 3,583,894). The hesperidine dihydrochalcone is of the formula

Jrn / 11/5/73

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OH

OH

II

subjected to the action of the enzyme system naringinase, its. ß-glucosidase activity was largely destroyed, so that it mainly only splits off the ehamnosis. The inactivation of the beta-glucosidase enzyme of the system is effected by heating an aqueous Naringinasesuspension to a temperature of about 60-65 ⁰ C for 30-120 minutes, at a pH from 6.4 to 6.8. However, this method is associated with a partial loss of bhamnosidase activity, the absolute value of which cannot be reproduced.

It has now surprisingly been found that when a glycolytic enzyme or enzyme system is fixed with Rhamnosidase activity, such as ITaringinase and represent hesperidinase, on a carrier, in particular on a solid activated carrier, the β-glucosidase activity is selectively inhibited.

The present invention accordingly relates to a process for the production of hesperetin dihydrochalcone glucoside from hesperidin dihydrochalcone using glycolytic enzymes or enzyme systems with rhamnosidase activity, as represented by hesperidinase and naringinase, which process thereby is characterized in that a carrier-bound enzyme or enzyme system with inhibited ß-glucosidase activity is used.

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The invention also relates to a glycolytisch.es enzyme or enzyme system derivative with rhamnoaidase activity and inhibited ß-glucosidase activity, which by coupling the enzyme or Enzyme system to a solid carrier material the ß-glucosidase activity, in particular with regard to hesperidine dihydrochalcone as substrate, has largely lost, as well as its production and use for converting hesperidin dihydrochalcone into hesperetin dihydrochalcone glucoside.

An enzyme or enzyme system preferred in connection with the present invention is hesperidinase, a commercially available product.

As solid support materials that can be used for manufacturing Enzyme preparations according to the invention, in particular hesperidinase Polymers with inhibited β-glueosidase activity are suitable on an organic and inorganic basis. As inorganic Materials come from natural and synthetic silicates and substances containing silicate, such as glass, sand, silicon gel, Diatomaceous earth, bentonite, wollastonite but also metal oxides such as aluminum oxide and hydroxyapatite are possible. Examples of suitable ones Organic-based polymers are cellulose and polyamides. In a particularly preferred embodiment In the present invention, porous glass can be used as the support material, such as is commercially available is, preferably with a particle size of about 80-400 mesh and an average pore diameter of about • 75 - 2000 1. The carrier material can, however, if the proportions require it to have a different shape and / or sizes.

The solid support material is expediently with a Layer of an aromatic diamine coated, which is advantageous by treatment with a solution of the amine,

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if necessary with slight warming, can be done. As the aromatic diamines, for example, 4,4'-diaminostilbene or 4,4'-diamino are 1,3-phenylenediamine, ^{4,4-diamino-l} diphenyl

diphenyl methane and · corresponding substituted compounds. A particularly preferred diamine is 4,4'-diamirio-diphenylmethane. The product obtained in this way is then diazotized in a manner known per se, e.g. by reaction with nitrite and acid, such as hydrochloric acid or acetic acid, expediently at temperatures between 0 ° and room temperature. Through the above described In the process, the carrier particles are given a firmly adhering coating that is used to fix the protein is activated.

In order to fix the enzyme to the activated solid carrier, it is treated with a solution of the enzyme, a protein / carrier ratio of 1-50 mg / g should expediently be maintained. Suitable solvents for the enzyme are all those familiar to the person skilled in the art, in particular buffer solutions, for example phosphate or citrate buffers. The reaction of the enzyme with the diazotized carrier material is preferably carried out in a buffer solution with a pH between about 4.5 and 8.0 with shaking from about 30 minutes to 24 hours, preferably from 1-2 hours, and at a temperature between 0 ° and 30 ⁰ C. particularly preferred in the case of using hesperidinase is a buffer of pH 6.0 to 7.5 and a Eeaktionstemperatur of about 4 ⁰ C.

The enzyme thus obtained, bound to a solid activated carrier, selectively inhibits its β-G-lucosidase activity is suitable for the degradation of hesperidin dihydrochalcone to hesperetin dihydrochalcone glucoside. In addition, owns the insolubilized enzyme obtained in this way has the known advantageous

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Adhesive properties: it can be easily separated from the substrate, for example by filtration or centrifugation, can be used several times and is particularly suitable for continuous litigation. Finally, the enzyme preparation obtained according to the invention has a high stability. For hesperidinase bound to porous glass, a particularly good activity and stability was found at continuous reaction in columns, as it is known, for example, from chromatography, observed.

The Hesperidin dihydrochalkons can be reacted with the carrier-bound enzyme in a manner known per se, both in stationary operation, for example in stirred reactors, or, as already mentioned, advantageously in continuous operation, for example in reaction columns. Solutions with a substrate concentration of about 5 ° are assumed, but solutions with a higher or lower concentration can of course also be used. The reaction product can be worked up without any problems and can be carried out in a known manner. For example, it can only consist of concentration to dryness without further purification, since the aglycone which may be obtained in small amounts as a by-product during the cleavage is also sweet and physiologically harmless.

The following examples illustrate the invention:

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example 1 Preparation of the activated carrier

1 g of porous glass (120-200 mesh, pore diameter: 2000 Å) was added to a solution of 200 mg of 4>4'-diaminodiphenylmethane in 10 ml of dry benzene. The mixture was heated on the water bath (80 °) for 5 minutes with occasional shaking, then suction filtered until all of the benzene was removed. The residue was poured into 20 ml of ice-cold water, 300 mg of sodium nitrite and then dilute hydrochloric acid (20 vol-fo) were added while stirring, up to a pH of 1-2. The mixture was then stirred for 10 minutes at 0 °, the pH was adjusted to 5 by adding 1N sodium hydroxide solution and the supernatant was decanted off. The residue was washed several times with ice-cold water, which had been brought to pH 4-5 by adding hydrochloric acid, until the supernatant was colorless. The particles thus obtained were used directly for the enzyme fixation.

Example 2 Fixation of the enzyme

1 g of the carrier material prepared according to Example 1 was added to 25 ml of an ice cold solution of 2 mg hesperidinase (5.2 units / mg) in 0.05 M phosphate buffer (pH 6.5-7.8) and shaken gently at 4 ° for 1 hour. It was then filtered, and the particles were washed three times with 50 ml of 1 M potassium chloride solution each time and washed once with 50 ml of 0.05 M citrate buffer (pH 6.5), suspended in 50 ml of 0.05 M citrate buffer pH 6.5 and then stored at 4 °.

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Example 3

Transferable; of hesperidindihydrochalkon in hesperetindihyd.rochalkongluc ο s id

2 g of hesperidinase (4.3 units) obtained according to Example 2 and bound to porous glass were shaken at 45 ° for 2 hours with a solution of 500 mg hesperidin dihydrochalcone in 20 ml 0.05'M citrate buffer (pH 4.5). Chromatographic analysis of the reaction solution showed that after this time the hesperidin dihydrochalcone had been converted quantitatively into hesperetin dihydrochalcone glucoside, traces of the corresponding aglycone being detected. The carrier-bound enzyme was filtered off with. 0.05 M citrate buffer (pH 6.5) washed at 45 ° and used again immediately or stored at 4 ° for renewed use. The piltrate was combined with the wash solutions and concentrated to dryness under reduced pressure. The residue was extracted three times with 50 ml of dioxane. After removing the solvent for 3 seconds, the extract gave 240 mg (yield 10fo) of the very sweet hesperetin dihydrochalcone glucoside.

An equally good yield of G-lucoside was obtained three times direct extraction of the reaction solution with ethyl acetate obtained without prior constriction.

By cooling the reaction solution with ice, a crystalline excretion of the glucoside is achieved. This work-up is not quantitative, but is well suited for larger batches where extraction methods are less suitable are.

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Example 4

Conversion of hesperidinedihydrochalcone. in hesperetin dihydrochalkODfflucoside

3 g of hesperidinase (8.1 units) obtained according to Example 2 and bound to porous glass were placed in a glass column (1.0 χ 10 cm) with a solution of 50 mg hesperidin dihydrochalcone in 0.05 M citrate buffer (pH 4.5) at 45 °, with a flow rate of 11 ml / h was maintained. Continuous chromatographic analysis showed the quantitative degradation of hesperidin dihydrochalcone to form hesperetin dihydrochalconium glucoside and rhamnose as well as a trace of aglycones.

Example 5

Conversion of hesperidine dihydrochalcone into hesperetin dihydrochalcone glucoside

In a reaction column which contained 5 g of carrier-bound hesperidinase (20 mg of enzyme) were in continuous operation over 2 weeks at an initial flow rate of 20 ml / h, which decreased to 10 ml / h towards the end of the operating time, 118 g of hesperidinedihydrochalcone hydrolyzed quantitatively without the enzyme's activity being exhausted.

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Claims (4)

■ - 9 - ι Claims X. Process for the preparation of hesperetin dihydro- chaUconglucosid from. Hesperidin dihydrochalcone by means of a glycolytic enzyme or enzyme system with rhamnosidase activity _f, characterized in that a carrier-bound enzyme or enzyme system with inhibited β-glucosidase activity is used. 2. The method according to claim 1, characterized in that nan a carrier-bound hesperidinase with inhibited .β-glueosidase activity used. 3. The method according to claim 2, characterized in that that the carrier is an activated silicate-containing material, 4. Process according to claims 2 and 3 * characterized in that » that the carrier consists of porous glass with a layer of a diazotized aromatic diamine. 5- The method according to claim 4, characterized in that the aromatic diamine is 4,4 ^f -diamino-diphenylinethane. 409835/1019